Bacillus is a genus of rod-shaped bacteria. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species. There are three important pathogenic members of Bacillus: B. cereus causes a foodborne illness similar to that of Staphylococcus; B. thuringiensis is an important insect pathogen, and is sometimes used to control insect pests; and B. anthracis causes anthrax in humans and animals. Under stressful environmental conditions, these cells shift to an alternative developmental pathway, sporulation, and produce oval endospores that can stay dormant for extended periods.
Much of what is known of the sporulation process comes from genetic studies of the nonpathogenic Bacillus subtilis. Unlike the pathogenic Bacillus spp. above, B. subtilis does not have an exosporium layer on its exterior. This outermost layer of the endospore consists of a basal layer surrounded by an external nap of hair-like projections (see FIG. 1). Filaments of the hair-like nap are predominantly formed by the collagen-like glycoprotein BclA, while the basal layer is comprised of a number of different proteins.
The use of bacteria spores as delivery systems has been demonstrated using a B. subtilis platform. See, Kim and Schumann, 2009. The B. subtilis platform was proposed as a vaccine delivery system using CotB and CotC fusion proteins. This platform displays a low level of C-terminally fused proteins on the spore coat of the endospores. Both CotB and CotC have complex structures with multiple cysteine bonds with other spore coat proteins and a rigid structure. The necessity to form multiple binding partners for Cot B/C incorporation hinders high protein expression levels on the surface of the spores. Additionally, the majority of the CotB and CotC proteins are in a complex 3D coat structure, with only a small percentage accessible for enzymatic reactions on the surface of B. subtilis spores. Therefore, a need remains for a delivery system capable of displaying a high percentage of MOIs on the exosporium for delivery and that allows substantial bioactivity of the MOIs.
The delivery system of the present invention solves the above-described problems. Specifically, the present invention provides a delivery system utilizing a small expression sequence that targets the MOIs to the most external portion of the exosporium surface allowing even the most complex MOI structures to freely fold in a native fashion for optimal bioactivity. Further, the present invention provides a delivery system with high expression of MOIs. The compositions, methods of making, and methods of using the delivery system of the present invention are described in detail below.